Elevated levels of high-mobility group A (HMGA) protein expression have been reported in almost every type of human cancer, including colorectal cancer, pancreatic cancer, and breast cancer. There are two forms of HMGA proteins, HMGA1 and HMGA2, which are encoded from two different genes. Both forms of HMGA are non-histone chromatin architectural transcription factors found broadly in eukaryotes. HMGA proteins are expressed at high levels in embryonic tissues during early development and at very low levels in normal differentiated somatic adult cells. Regulation of gene expression is a primary function of HMGA in these cells and HMGA proteins are involved in both positive and negative regulation of genes responsible for apoptosis, cell proliferation, immune response and DNA repair. Overexpression of HMGA has been shown to increase cell proliferation contributing to tumor growth.
In addition, it has been shown that HMGA1 interacts with the p53 tumor suppressor protein and inhibits its apoptotic activity. It has also been shown that high expression levels of HMGA1 are responsible for chemotherapy resistance in pancreatic cancer cell lines and that suppression of HMGA1 expression by siRNA restored the cells sensitivity to gemcitabine. HMGA2 is responsible for maintaining Ras-induced epithelial-mesenchymal transition that promotes tissue invasion and metastasis. Down regulation of overexpressed HMGA2 has been shown to inhibit cell proliferation in human pancreatic cancer cell lines. While the precise role that HMGA plays in cancer is not yet completely understood, HMGA has been suggested as a potential biomarker for tumor progression and is a drug target for cancer therapy development.
An early structural study showed that HMGA does not adopt a conventional protein structure composed of a helices or β sheets but rather binds in the minor groove of AT-rich double-stranded DNA through crescent-shaped DNA binding motifs referred to as “AT-hooks.” In contrast to classical transcription factors that bind specific DNA sequences, HMGA acts as an architectural transcription factor that binds a specific type of DNA structure, i.e. the minor groove of A:T tract DNA. Due to this unique DNA binding property of HMGA, several cancer therapy drugs, such as FR900482 and FL317, have been designed as competitive HMGA1 inhibitors that bind to the minor groove of AT-rich DNA. These drugs however, have shown high toxicity in humans. Recently, it has been shown that Spiegelmer NOX-A50 is a potent inhibitor of HMGA1 activity and proposed the use of artificial HMGA1 substrates that block HMGA1 binding to its natural DNA substrate. In principle, decreasing all HMGA protein activity could result in inhibition of unwanted cell proliferation and reestablishment of apoptosis, reducing cancer progression.
Nucleic acid ligands designed or selected to inhibit the activity of pathogenic proteins are referred to as aptamers or “decoys”. Nucleic acid aptamers contain variable sequences and/or modified chemical structures to facilitate binding to their protein targets with high specificity and an equal to, or higher, affinity compared to their unmodified oligomer counterparts. They are widely studied for biotechnological and therapeutic applications because they have little or no immunogenicity compared to antibodies and several applications have been reported. For example, one study has shown that overexpression of a 60-nucleotide RNA decoy used as a antiviral treatment showed inhibition of Tat-mediated HIV replication in vitro by 90%. In another study, a 2′-fluoropyrimidine RNA was designed as a vascular endothelial growth factor inhibitor that reduced lung metastasis in mice. A DNA aptamer targeting transcription factor E2F, which is essential in cellular proliferation regulation, was shown to decrease cell proliferation in vascular smooth muscle cells.
In addition to engineered specificity, an important property of DNA aptamers is that they are sometimes designed to be resistant to endogenous nuclease activity in vivo. For example, both phosphorothioate DNA (sDNA), which contains sulfur substituted for one oxygen atom in the phosphodiester backbone, and phosphorodithioate DNA, which contains sulfur substitution of two oxygen atoms in the phosphodiester backbone, have been shown to have shown increased resistance to nuclease S1 and Deoxyribonuclease I (DNase I) activity as the number of sulfur substitutions increases.
Since down regulation of both HMGA1 and HMGA2 proteins contributes to the inhibition of tumor growth, the strategy of targeting both HMGA1 and HMGA2 may result in a potentially more potent therapeutic strategy than targeted inhibition of either protein alone. Therefore, there is a need for nuclease resistant DNA aptamers that both inhibit pancreatic cancer tumor growth and increase the sensitivity of pancreatic cancer cells to chemotherapy by down regulation of both HMGA1 and HMGA2 proteins and are resistant to endogenous nuclease attack.